Purpureocillium lilacinum for plant growth promotion and biocontrol against root-knot nematodes infecting eggplant

Purpureocillium lilacinum is a biocontrol Ascomycota fungus against various plant pathogens. In the present study, the efficacy of P. lilacinum was evaluated against a root-knot nematode, Meloidogyne incognita that infects eggplants. We performed an in vitro experiment in which the direct effects of P. lilacinum on the second-stage juvenile survival and egg hatching of M. incognita were tested at different exposure times. The results showed that P. lilacinum significantly reduced the rates of egg hatching and juvenile survival in a dose-dependent manner. Microscopic observation demonstrated that P. lilacinum directly penetrated the eggs and contacted the juveniles, indicating how P. lilacinum parasitizes M. incognita. We also performed a pot assay in which soil-grown eggplants were treated with P. lilacinum followed by inoculation with M. incognita. The results indicated that P. lilacinum effectively reduced the nematode population and the number of galls in plant roots. Interestingly, application of P. lilacinum resulted in significant enhancements in plant growth and biomass, even under nematode infection, while it improved plant photosynthetic pigments, i.e., chlorophyll and carotenoids. Taken together, our study suggested that P. lilacinum can be used as a plant growth-promoting fungus and a biological nematicide for disease management of root-knot nematodes in eggplants.


Introduction
Eggplant, Solanum melongena L., also known as brinjal in Indian subcontinents or aubergine in Europe, belongs to the family Solanaceae and is one of the most important vegetable crops. Eggplants are susceptible to many diseases including soilborne diseases causing great losses in yield and quality. In general, diseases caused by soilborne pathogens are difficult to control, and plant resistance to many soilborne pathogens has not been fully studied [1][2][3]. Plant-parasitic nematodes are also not an exception to soilborne pathogens, causing high losses in the agriculture sector for eggplants [4].
Root-knot nematodes, Meloidogyne spp., are soil-borne sedentary endoparasites and are one of the most important plant-parasitic nematodes. Root-knot nematodes decrease crop yield by up to 75% in the heavily infested fields [5,6]. The nematodes invade the plant roots with the help of stylet and migrate intercellularly and develop the feeding site where they initiate the formation of giant cells known as galls or knots [7]. Generally, chemical pesticides and soil fumigation are used for plant disease management on a large scale in the agriculture sector. Heavy use of toxic chemicals causes human health issues and environmental toxicity. To reduce the use of synthetic chemicals, it is urgently needed to develop alternative control methods that have minimal impact on the environment. Over recent years, biocontrol methods and biotechnological approaches have been widely studied and applied for disease management caused by plant-parasitic nematodes [8,9].
Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is a nematophagous fungus that belongs to the Ascomycota phylum [10,11]. This fungus occurs naturally in the soil and rhizospheres of many crops [12,13]. It can grow at a wide range of temperatures (8-38˚C) and pH tolerances [14,15]. Purpureocillium lilacinum forms a dense mycelium from which conidiophores arise. Spores germinate when suitable conditions, e.g., moisture and nutrients are met [14,16]. This soil fungus has been extensively tested for the biocontrol of plant-parasitic nematodes [17,18]. It has also been reported as an effective biocontrol of different species of insects, e.g., cotton aphids, western flower thrips, glasshouse red spider mites, greenhouse whiteflies, and leaf-cutting ants [19][20][21]. Recently, the fungus was evaluated as a biocontrol agent against some fungal plant pathogens, such as Sclerotinia sclerotiorum, Verticillium dahliae, Phytophthora capsici, and P. infestans [22][23][24]. These reports suggest that P. lilacinum is one of the most promising biocontrol agents against plant pathogens and pests.
In the present study, the efficacy of P. lilacinum was evaluated for plant protection against M. incognita, which causes root-knot disease in eggplants. Based on in vitro experiments and greenhouse trials, our data demonstrated that P. lilacinum significantly alleviates infection of the root-knot nematode and enhances eggplant growth, suggesting that P. lilacinum is a useful biocontrol agent for disease management of root-knot nematodes in eggplants.

Fungal culture and inoculum preparation
Purpureocillium lilacinum was obtained from the Indian Type Culture Collection at the Indian Agriculture Research Institute (New Delhi, India). Culturing of P. lilacinum was performed as described in our previous study [25]. Subculturing of fungus was performed on potato dextrose agar (PDA) medium for use in the experiment. Richard's liquid medium was used to obtain the inoculum of the fungus [26], which contained 10 g/L of potassium nitrate, 5 g/L of potassium dihydrogen phosphate, 2.5 g/L of magnesium sulfate, 0.02 g/L of ferric chloride, and 50 g/L of sucrose. Each 80 mL of the Richard's liquid medium was placed in Erlenmeyer flasks followed by sterilization at 103.4 kPa for 15 min in an autoclave machine. The flasks were incubated at 25 ± 1˚C for~15 days on a incubator shaker with 100 rpm (Fig 1). For inoculum preparation, the fungal mycelia mat on filter paper was washed in sterile water, and extra water and nutrients were removed with blotting paper. Ten grams of mycelia mat (fungal inoculum) was mixed in 100 mL of distilled water followed by blending in a Waring blender (10,000 RPM) for 30 s. The inoculum collected was adjusted to 10 8 CFU/mL and labeled as a standard suspension (S), and consecutive concentrations, such as S/2, S/10, S/25, and S/50, were prepared using distilled water. Ten milliliters of standard (S) suspension were used to inoculate eggplants in a pot assay.

Propagation of Meloidogyne incognita
The root-knot nematode M. incognita was isolated from infected eggplant roots and maintained in greenhouse for research purpose [27]. Egg masses were hand-picked with the help of sterilized forceps from infected roots. The isolated egg masses were washed with distilled water and placed in a small sieve of 9-cm diameter with 1-mm pore size containing layers of tissue paper. The sieve was placed in a Petri plate containing distilled water deep enough to contact the egg masses and these assemblies were kept in an incubator at 25 ± 1˚C for the hatching of second-stage juveniles (J2). Hatched J2 were collected and the volume of the nematode suspension was adjusted to 200 ± 5 nematodes per mL. Twenty mL of the nematode suspension (4000 freshly hatched J2) was added to each pot [27]. The plants were harvested 90 days postinoculation, and the roots were cut into 4-5 cm pieces after washing to collect eggs. The eggs were hatched in water, and active J2 were collected (Fig 2).

Mortality bioassay
To measure the efficacy of P. lilacinum on the mortality of M. incognita, 10 mL of fungal suspension with different concentrations (S, S/5, S/10, S/20, S/50, S/100) was placed in each empty Petri dish. Then, 100 freshly hatched J2s were added to each Petri dish. The dishes were

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incubated at 25 ± 1˚C in a biochemical oxygen demand (BOD) incubator, and the effect on mortality was observed after 48 h intervals.

Egg hatching assay
To perform the egg-hatching assay, 10 mL of fungal culture suspension of different concentrations (S, S/5, S/10, S/20, S/50, S/100) was placed in each Petri dish. Three eggs masses of the same size were placed in each Petri dish. The rates of nematode hatching were observed after incubation at 27-28˚C for 48 h by counting the number of hatched juveniles under the microscope. Each treatment was replicated five times at room temperature.

Microscopic observation of the effect of the fungi on nematode eggs and juveniles
Purpureocillium lilacinum was incubated in the center of a Petri dish containing PDA medium amended with streptomycin at 10 mg/L and incubated at 25 ± 2˚C for 10 days. After incubation, 100 surface-sterilized M. incognita eggs were placed on the dish (the eggs were sterilized with 2% sodium hypochlorite solution for 5 minutes beforehand). After 10 days, the eggs were stained with cotton blue, and the percentage of egg parasitism was evaluated by counting the parasitized and non-parasitized eggs under a microscope. The eggs with either direct hyphal penetration or disintegration of their contents were considered dead [28], while eggs that contained live juveniles and eggs hatching juveniles were counted as viable.

Inoculation of P. lilacinum with M. incognita-infected eggplants in a greenhouse
A greenhouse experiment was conducted to evaluate the antagonistic properties of P. lilacinum against the root-knot nematode M. incognita infecting eggplants. The eggplant seeds (cv. VNR-218) were obtained from VNR Nursery Pvt. Ltd. (Chhattisgarh, India). Before sowing, eggplant seeds were surface sterilized with 0.1% sodium hypochlorite solution for 1 min and then washed with distilled water. Two-week-old eggplant seedlings were planted in plastic pots (30-cm diameter) containing 3.5 kg of sterilized sandy loam soil collected from the field of A. M.U. (Aligarh, India). The collected soil was autoclaved at 137.9 kPa for 20 min. Inoculum of P. lilacinum (10 8 CFU/mL) was applied at 10 mL/pot as a soil drench. Second-stage juveniles of M. incognita (4000 J2s/pot) were added one day after the fungal inoculation. Three treatments of pots were prepared with five replicates for each treatment, including a control, as follows: C, control = no fungi and nematodes; M, inoculated with M. incognita; and M+P, inoculated with M. incognita and P. lilacinum.

Measurement of plant growth and disease evaluation
Plants were collected 90 days after nematode inoculation. The plant growth criteria, including plant height, plant weight, and shoot dry weight, were measured. Plants were uprooted carefully. Plant height was measured in cm from the end of the root up to the top of the first leaf.
To determine the plant's fresh weight, excess water was removed by blotting before weighing. Plants were kept in envelopes at 60˚C for 2-3 days before dry weight determination. The roots were washed gently with tap water to separate the soil particles and were stained with acid fuchsin in lactic acid to evaluate the phases of the nematode developmental stages, females, galls, and egg masses under binuclear conditions [29]. Juveniles of nematodes were extracted from the soil using a modified Baermann technique and counted using a stereoscopic microscope with a counting slide [30]. Two hundred fifty grams of subsampled well-mixed soil from each treatment was processed by Cobb's sieving and decanting technique followed by Baermann funnel extraction to obtain nematodes [31]. Every 24 h, the nematode suspension was collected, and the numbers of nematodes were counted in five aliquots of 1 mL of suspension from each sample. The population of nematodes per kg of soil was calculated from the means of five counts. For the estimation of photosynthetic metabolites in plant leaves, the chlorophyll and carotenoid contents were measured based on a previously published method [32].

Data analysis
Sigma Plot was used for the preparation of graphs of the mean value with the standard error. All experiments were repeated at least five times, and the data obtained were analyzed using ANOVA followed by the Tukey-Kramer multiple comparison test. A difference with P < 0.05 was considered significant.

Direct effects of P. lilacinum on egg hatching and juvenile viability of M. incognita under in vitro conditions
Direct exposure of M. incognita to P. lilacinum was performed to test whether there were any inhibitory effects on egg hatching and juvenile viability of M. incognita. To this end, the eggs and J2s of M. incognita were incubated with suspension cultures of P. lilacinum at different concentrations with serial dilution (S, S/5, S/10, S/20, S/50, S/100), where S is 10 g mycelia in 100 mL distilled water. As shown in Fig 3, P. lilacinum showed high inhibitory activities against M. incognita, while the effects became more prominent upon increasing the exposure time and concentration. Egg hatching was reduced as the concentration of P. lilacinum increased. Maximum hatching occurred after 48 hours, and the highest inhibition of egg hatching was observed at the (S) standard concentration with 62.8% less than control in hatching (Fig 3A). The concentration of S/5 caused 52.5%, S/10 caused 38.4%, S/20 caused 30.7% and S/50 caused a 17.9% reduction in hatching after 48 h (Fig 3A). Regarding the effect of P. lilacinum on nematode viability, the highest mortality (61%) was observed in the standard (S) suspension of P. lilacinum (Fig 3B), indicating that juvenile mortality was greatly influenced by the concentration of fungal inoculum. The lowest mortality (31%) was observed at the S/50 concentration, which was the lowest fungal concentration we tested (Fig 3B). These results demonstrated that P. lilacinum directly inhibited the egg hatching and the juvenile viability of M. incognita.

P. lilacinum parasitizes eggs and juveniles of M. incognita
We then observed the penetration and infection of fungi (Fig 4). Egg parasitism was recorded after ten days of fungal exposure to eggs, and the presence of mycelium and spores inside or outside the eggs was considered to indicate infection. The fungi were also observed penetrating hatching eggs (Fig 4B and 4C). After ten days, almost all eggs were found infected by fungi. Additionally, the fungal hyphae directly interacted with and reduced the juveniles' mortality ( Fig 4D). These results demonstrated how P. lilacinum parasitizes the eggs and juveniles of M. incognita.

Effect of P. lilacinum on plant growth of pot-grown eggplants
The inoculation of M. incognita ("M" in Fig 5) inhibited the growth of the soil-grown eggplants; for example, there was a 19.7% reduction in plant height and a 23.8% reduction in plant fresh weight. Significant reductions in plant biomass, for example, a 12.2% reduction in shoot dry weight and a 9.1% reduction in root dry weight, were also found after the inoculation of M. incognita compared to the uninoculated plants ("C" in Fig 5). In contrast, the application of P. lilacinum ("P+M" in Fig 5) mitigated the adverse effects caused by M. incognita infection and further enhanced plant growth in terms of height and biomass (Fig 5A-5D). In the same manner, P. lilacinum significantly improved the chlorophyll and carotenoid contents in plants (Fig 6A and 6B). This result suggests that P. lilacinum can be used as a plant growth-promoting fungus as proposed previously [8]. Finally, we found that the application of P. lilacinum caused significant reductions of 62% and 52% in the gall numbers and the nematode population, respectively (Fig 6C and 6D). Transverse sections of the eggplant roots showed that the presence of female M. incognita was less frequently observed when P. lilacinum was preinoculated onto the eggplant roots (Fig 7). Taken together, our results suggested that P. lilacinum, which directly acts on M. incognita, can be useful as an effective nematicide to protect eggplants against root-knot nematodes.

Discussion
The present study investigated the biocontrol fungus P. lilacinum for plant protection against plant-parasitic nematodes. This fungus is one of the biocontrol agents approved for use against nematodes in farms and commercial fields. However, information regarding its efficacy is scarce in different plants, e.g., eggplants.  No inoculation of either M. incognita orP. Lilacinum was used as a control as labeled "C". Plants were collected 90 days after nematode inoculation to measure total chlorophyll (A) and carotenoids (B) as well as the nematode population (C) and the numbers of galls per root (D). Different letters indicate statistically significant differences at P < 0.05 (n = 5) and "n.d." represents "not detected". https://doi.org/10.1371/journal.pone.0283550.g006

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Purpureocillium lilacinum employs flexible lifestyles, such as soil saprobes, plant endophytes, and nematode pathogens. The fungus was proposed as a nematode egg-pathogenic fungus, but several papers have demonstrated that it infects all life stages of root-knot nematodes [33,34]. The timing of fungal application has also been studied. For example, Dahlin et al. recommended a sequential application of a chemical pesticide (e.g., fluopyram) followed by P. lilacinum application since it was highly effective to control the nematodes and increasing the crop yields in comparison to a single application of one or other of two [35]. Currently, the fungus is commercially used as a biological nematicide (e.g., MeloCon, by Certis USA LLC) against a wide range of nematodes, including root-knot, cyst, burrowing, reniform, root lesion, and false root nematodes [36]. Purpureocillium lilacinum is getting popular since it is more cost-effective and environmentally sound when compared to synthetic nematicides [37].
In the present study, we observed based on the in vitro experiment that P. lilacinum directly penetrated hatched eggs and juveniles of M. incognita, which are infectious for eggplants. The data showed that P. lilacinum reduced at most 62.8% and 61% in the egg hatching rate and the juvenile mortality rate of M. incognita, respectively. Our results were comparable with the previous study using two different strains of P. lilacinum (local strain PLA and PLB and commercial strain PLM) that exhibited 66.0-78.8% parasitism on eggs and 88-89% reduction in the egg hatching [38]. Our microscopic observation demonstrated that P. lilacinum directly penetrated the eggs and contacted the juveniles, indicating how the fungus parasitizes M. incognita. These results demonstrated how the fungus breaks into the nematode body to parasitize or directly kill and how the fungus reduced the rates of egg hatching and juvenile survival.
We demonstrated that a preinoculated soil with P. lilacinum reduced M. incognita root galling by 52% and egg masses by 62% on eggplants. These data in eggplants were comparable with that seen in tomato for which Kiewnick et al. [39] reported 45% reduction in the galling index and 69% reduction in the number of egg masses per root system after preinoculation with P. lilacinum strain 251 (another commercial strain). Taken together, P. lilacinum is the effective biocontrol agent against M. incognita infecting various plants, including eggplants.
What could be the possible molecular mechanisms by which P. lilacinum parasitizes M. incognita? The genomic sequence of P. lilacinum revealed enriched contents of carbohydrateactive enzymes (CAZymes), proteases, secondary metabolites, and pathogenesis-related genes in comparison to other fungi [40]. In particular, it has been demonstrated that cuticle-degrading protease, chitinase, and serine protease, play important roles in degrading nematode eggshells [41][42][43] and thereby P. lilacinum parasitizes M. incognita. In addition, another studies reported that the proteins with CFEM (common in fungal extracellular membrane) domains were up-regulated during the infection of nematode eggs and suggested that the CFEM proteins is important for the recognition of nematode-eggs by the fungus [44,45]. Recently, P. lilacinum was shown to produce secondary metabolites (e.g., leucinostatins, paecilomide, and acremoxanthones) that cause strong mortality and inhibit nematode reproduction. These metabolites could be another mechanism that kills nematodes [46][47][48].
Our data demonstrated that P. lilacinum promotes plant growth in eggplants. This positive effect could be because P. lilacinum alleviated the adverse effect of the nematode infection on the plant growth. However, P. lilacinum has been shown to have beneficial effects on maize, common bean, and soybean plants, where nitrogen and phosphate availabilities were increased in soil [13]. Interestingly, our data showed that P. lilacinum promoted the accumulation of photosynthetic pigments, i.e., chlorophyll and carotenoids, which could be attributed to increased nutrient availability in the soil, as reported previously [13].
In conclusion, P. lilacinum is an effective biocontrol agent against the root-knot nematode M. incognita infecting eggplants, while it provides plant growth promotion. The fungus has been well studied in terms of its molecular and ecological characteristics. In the future, in-depth studies of the direct beneficial effects of the fungus on plant physiology and immune responses will be needed. This type of research is important because a lack of knowledge of how this biocontrol agent works on plants may deprive growers of a potentially effective application strategy. In addition, some strains of P. lilacinum could pose a medical hazard affecting on human and animal health due to its potential mycoses of the lungs, heart, eyes, and skin, e.g., hyalohyphomycosis [49]. Although some P. lilacinum strains can be pathogenic to animals, including humans, the risk of infection through the current mode of application is likely to be very low. Nevertheless, it is always important to conduct thorough risk assessments to ensure the safety of human health and the environment, as is the case with other biocontrol agents and biopesticides [50].